The stratum corneum of the epidermis represents an important interface between the external and the internal environments of vertebrates and its integrity is essential for survival. The main functional component of this layer is the cell envelope (CE). The CE is a specialized composite structure that replaces the plasma membrane of terminally differentiating keratinocytes. It consists of proteins cross-linked by covalent bonds into a rigid envelope with lipids covalently attached to its external surface and the keratin filament/matrix complex interacting with its internal surface. The cross-links between proteins are disulfide bridges, and more importantly covalent cross-links between glutamine and lysine residues, catalyzed by transglutaminases (TGases). A major protein component of the CE has been recently characterized and it has been named loricrin. Loricrin comprises greater than 50% of the protein in CE; therefore, it is predicted that its synthesis is critical for formation of a functional CE, and thus the integrity of the skin. Since mutations in keratinocyte transglutaminase (TGK) cause lamellar ichthyosis (LI), a severe congenital skin disorder characterized by generalized large scales and variable redness, it is hypothesized that the failure to synthesize loricrin, the most abundant substrate of TGK, will result in the development of a phenotypically similar disease. The overall goals of this proposal are to understand how expression of the loricrin gene is regulated and to determine the contribution of loricrin to the formation and maintenance of epidermal barrier function. Both in vivo transgenic and in vitro tissue culture techniques will be used to explore regulation of the loricrin gene. The transgenic experimental system will be utilized to identify those elements required for correct tissue, developmental and differentiation-specific expression. The micro analysis of these elements will be examined by insertion of putative regulatory regions into reporter constructs, followed by transfection with primary epidermal keratinocytes. Proteins that interact with these elements will be characterized, and those that are unique to keratinocytes will be cloned. To determine the role of loricrin in formation and function of the CE, mutant mice will be created, via homologous recombination in embryonic stem cells, in which the loricrin gene has been deleted. The yeast two-hybrid system, as well as alternative strategies will be used to identify domains of loricrin that interact with other cellular proteins, and to confirm their functional importance, mutations will be engineered into these regions and assessed in transgenic mice. The experimental approach to ablate the loricrin gene will define the contribution of loricrin to epidermal barrier function, and the identification of function domains of loricrin will provide insight into how components of the composite CE interact and function. Results from these studies, it is anticipated, will not only indicate possible diseases in which loricrin mutations are causal, but will also be applicable to a variety of diseases in which patients exhibit a defective barrier function, manifested in part by dry skin symptoms.